The objective of this proposal is to examine the role of phosphorylation in rat neurofilament (NF) expression and assembly during development in 2 different rat model systems: 1) An in vivo system consisting of normal rats at different stages of development, and 2) An in vitro system comprised of PC12 cells (a clonal cell line derived from a rat pheochromocytoma) and their normal counterpart, i.e., primary cultures of rat sympathetic neurons. PC12 cells are selected for comparison because they express aberrant NF proteins which may be due to abnormalities in phosphorylation. Using an existing library of monoclonal antibodies (MAs) to bovine phosphorylated and dephosphorylated NF triplet proteins, non-phosphorylated ("nascent") rat NF subunits will be isolated and characterized immunochemically and immunohistochemically in vivo and in vitro. A second generation of MAs to phosphorylated, non-phosphorylated ("nascent") and dephosphorylated rat NF subunits will be prepared and characterized. Such MAs are essential reagents for elucidating the mechanisms regulating NF subunit phosphorylation and assembly. Specifically, during development, the distribution of phosphate-independent and phosphate-dependent epitopes will be localized; the sites and states of phosphorylation will be identified; the effect of phosphorylation on assembly of NF subunits will be examined; and the activities of protein kinase(s) will be correlated with states of NF subunit phosphorylation. The proposed studies will probe fundamental aspects of NF expression, phosphorylation and assembly. They will also illuminate possible mechanisms of abnormal NF expression during development.